RhoE belongs to the Rho GTPase family, the members of which control actin cytoskeletal dynamics. RhoE induces stress fiber disassembly in a variety of cell types, whereas RhoA stimulates stress fiber assembly. The similarity of RhoE and RhoA sequences suggested that RhoE might compete with RhoA for interaction with its targets. Here, we show that RhoE binds ROCK I but none of the other RhoA targets tested. The interaction of RhoE with ROCK I was confirmed by coimmunoprecipitation of the endogenous proteins, and the two proteins colocalized on the trans-Golgi network in COS-7 cells. Although RhoE and RhoA were not able to bind ROCK I simultaneously, RhoE bound to the amino-terminal region of ROCK I encompassing the kinase domain, at a site distant from the carboxy-terminal RhoA-binding site. Overexpression of RhoE inhibited ROCK I-induced stress fiber formation and phosphorylation of the ROCK I target myosin light chain phosphatase. These data suggest that RhoE induces stress fiber disassembly by directly binding ROCK I and inhibiting it from phosphorylating downstream targets.
We have investigated the role of the small guanosine-trisphosphate (GTP)-binding proteins, Rho, Rac, and Cdc42, in the early responses of human umbilical vein endothelial cells (HUVECs) to TNF-alpha (tumor necrosis factor-alpha). Quiescent confluent HUVECs incubated with TNF-alpha for 5-30 min showed an increased formation of membrane ruffles, filopodia, and actin stress fibres followed by cell retraction and formation of intercellular gaps. This process was accompanied by the dispersion of cadherin-5 from intercellular junctions. TNF-alpha also induced a transient increase in polymerized F-actin, as determined both by measuring G-actin content and by quantifying fluorescent emission from fluorescein isothiocyanate (FITC)-phalloidin-labelled F-actin. Microinjection of cells with activated RhoA protein led to an increase in polymerized actin, formation of stress fibres, cell retraction as well as dispersion of cadherin-5. The proteins Cdc42 and Rac induced qualitatively similar effects to Rho, although not as dramatic and in addition induced formation of filopodia and lamellipodia. Microinjection of cells with a Rho inhibitor, C3 transferase, prevented gap formation caused by TNF-alpha. Similar effects were observed in cells microinjected with the dominant inhibitory proteins N17Cdc42 and N17Rac1. Cell retraction and gap formation were also prevented by inhibitors of myosin light chain kinase (MLCK). Our data suggest that Cdc42, Rac, and Rho are activated in a hierarchical cascade following stimulation with TNF-alpha leading to actomyosin-mediated cell retraction and formation of intercellular gaps.
Cdc42 induces β1 integrin expression at the transcriptional level via the transcription factor SRF to promote cancer cell interaction with endothelial cells.
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